Friday, February 26, 2010

Luminex Technology



We discussed a review article during our transplant clinic morning session about Luminex technology for HLA antibody detection in organ transplantation published in Nephrology 2009; and would like to share it with you.

Pre-formed donor-specific human leukocyte antigen (HLA) class 1 antibodies (DSA) in renal transplant recipients causes hyperacute rejection; there has been an imperative to test all potential recipients prospectively for HLA antibodies to avoid transplanting incompatible grafts.

Since was first developed until recently, the method universally used for antibody detection was the complement-dependent cytotoxicity (CDC) assay. In recent years, solid phase assays have been introduced as methods for HLA antibody screening which have again redefined the definition of pre-sensitization. Luminex is a solid phase assay which consists of a series of polystyrene microspheres (beads), containing fluorochromes. Test is run as shown in the Figure.

Another important concept is PRA (Percent Reactive Antibody); it is the percentage of cells in a random panel which contains all known HLA types; giving positive results with anti-serum. PRA is useful in estimating the probability of a patient receiving a crossmatch-negative kidney.

By referring to this review study, the comparison and advantages of Luminex over CDC:
1-CDC checks for cell death caused by complement fixing antibodies against any cell antigen, while DSA by Luminex checks for the presence of the antibody against HLA type ONLY.
2-Luminex DSA is much more sensitive and can detect Donor-Specific HLA antibodies which in some cases associated with rejection in the presence of negative donor CDC cross-match. Large studies by Gibney et al and Smith et al indicated that a further subset of patients with DSA detectable only by Luminex is at high risk of graft rejection
3-CDC method can detect any Ab directed at the cell surface of a B or T cells; HLA and non-HLA antibodies and these antibodies are complement dependent in causing cell death. Luminex on the other hand detects ONLY the HLA antibodies itself, it doesn’t discriminate between complement fixing or non-compliment fixing HLA-Ab.

The question is: whether those non-compliment fixing Ab are detrimental to the graft and, if not, are we excluding patients from receiving compatible graft by using Luminex tech.
As per Washerman et al C4d+ “complement fixing Ab”, Flow + “Luminex positive” HLA class 1 antibodies graft survival is inferior to C4d -, flow + and flow – patients.

Now, what about those non-compliment fixing antibodies that are detected by Luminex, do they have an effect on the graft?
Smith et al showed that 1 year survival of renal graft of patients with C4d+ DSA was 20%, C4d – DSA was 54%, C4d + non-DSA patients was 91%. The interesting observations are the profound negative effect of the compliment fixing antibody on graft survival and also the reduction in graft survival of the C4d- DSA+

Other significant benefit of Luminex is detecting HLA-class 2 Ab in pre- and post-transplant with high level of sensitivity.

Having the fact that Luminex is far more sensitive than CDC, and at least in many cases detects additional clinically relevant Ab, the question is Can Luminex HLA replace CDC?
Also, keep in mind that on the other hand some patients with CDC-neg and Luminex positive DSA had uneventful post transplant course without evidence of rejection! So, having too much sensitive test can potentially contribute to denying some patients a suitable Graft?!

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